Gata1^low BMMCs are characterized by high proliferation rates and generate mast cell precursors as early as day 7, but the cells remain partially immature up to day 26. (A) BMMCs from Gata1^low mice generate more cells and for longer time than BMMCs from wild-type littermates and W/W^v mice. Time-course analysis of BMMCs seeded with marrow cells from wild-type, Gata1^low, and W/W^v mice are presented in the top, middle, and bottom panel, as indicated. The dotted lines indicate the regular demipopulation necessary to feed the cultures. Results are presented as mean (± SD) of at least 6 independent cultures per experimental animal. Asterisk indicates values statistically different (P < .001) between BMMCs seeded with Gata1^low and wild-type, or W/W^v cells. (B) Representative flow cytometric analysis for c-Kit and FcϵRI expression of cells obtained in BMMCs seeded with marrow from wild-type, Gata1^low, and W/W^v mice, as indicated. Cells were analyzed either at day 7, 21, or 26 of culture. Negative controls were represented by cells labeled with irrelevant antibodies and are not presented for convenience. Similar results were obtained in at least 3 independent experiments per group of mice (see panel C). (C) Frequency of c-Kit^high (on the left) and FcϵRI⁺ (as percent of c-Kit^high, on the right) cells in BMMCs seeded with bone marrow from wild-type (large hatched bars), Gata1^low (⊡), and W/W^v (tight hatched bars) mice. Cells were analyzed either at day 7, 21, or 26 of culture, as indicated on the x-axes. Results are presented as the mean (± SD) of at least 3 independent experiments per group of mice. Values observed in Gata1^low BMMCs and statistically different from those obtained in the corresponding cultures with wild-type and W/W^v cells are indicated with a single asterisk (P < .05) and double asterisks (P < .01), respectively. (D). Semiquantitative RT-PCR analysis for the expression of mast cell specific genes (MMCP-6, MC-CPA, and MITF) as well as of Gata1, Gata2, and NFE2 in cells obtained after 26 days in BMMCs seeded with marrow from wild-type, Gata1^low, or W/W^v mice, as indicated. The Gata1^low c-Kit^high cells were divided into immature (FcϵRI⁻) and mature (FcϵRI⁺) cells by sorting (> 95% purity on reanalysis, not shown). c-Kit^highFcϵRI⁻ cells from wild-type and W/W^v BMMCs could not be isolated because of their infrequency. The triangle on top of the panels indicates increasing numbers of cycles. Similar results were observed in 2 additional experiments per experimental group; n.d. indicates not done. (E) Time-course analysis of serotonin uptake (on the top) and release (on the bottom) by cells obtained in BMMCs from wild-type (large hatched bars), Gata1^low (⊡), and W/W^v (tight hatched bars) mice. Cells were analyzed either at 7, 21, or 26 days of culture, as indicated on the x-axes. Levels of serotonin are expressed as cpm/10⁵ cells. The total serotonin incorporated was measured by lysing BMMCs in Triton. Serotonin release was induced by IgE-αIgE stimulation. Positive and negative controls were represented by cells stimulated with IgE alone, medium + αIgE; medium + HSA + DNP and ionomycin and are not shown for clarity. Results are presented as the mean (± SD) of at least 3 separate experiments, per group of mice, performed in duplicate. Values observed in Gata1^low BMMCs and statistically different from those obtained in the corresponding cultures with wild-type and W/W^v cells are indicated with a single asterisk (P < .05) and double asterisks (P < .01), respectively.